Direct binding of GABAA receptor β2 and β3 subunits to gephyrin

GABAergic transmission is essential to brain function, and a large repertoire of GABA type A receptor (GABA_AR) subunits is at a neuron's disposition to serve this function. The glycine receptor (GlyR)‐associated protein gephyrin has been shown to be essential for the clustering of a subset of GABA_AR. Despite recent progress in the field of gephyrin‐dependent mechanisms of postsynaptic GABA_AR stabilisation, the role of gephyrin in synaptic GABA_AR localisation has remained a complex matter with many open questions. Here, we analysed comparatively the interaction of purified rat gephyrin and mouse brain gephyrin with the large cytoplasmic loops of GABA_AR α1, α2, β2 and β3 subunits. Binding affinities were determined using surface plasmon resonance spectroscopy, and showed an ~ 20‐fold lower affinity of the β2 loop to gephyrin as compared to the GlyR β loop–gephyrin interaction. We also probed in vivo binding in primary cortical neurons by the well‐established use of chimaeras of GlyR α1 that harbour respective gephyrin‐binding motifs derived from the different GABA_AR subunits. These studies identify a novel gephyrin‐binding motif in GABA_AR β2 and β3 large cytoplasmic loops.     

Differential localization of GABAA receptor subunits within the substantia nigra of the human brain: an immunohistochemical study

Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.     

Early continuous white noise exposure alters l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor subunit glutamate receptor 2 and γ‐aminobutyric acid type a receptor subunit β3 protein expression in rat auditory cortex

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate‐level noise delayed the emergence of adult‐like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical‐period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA_A receptor subunit β3 in the auditory cortex after noise rearing. Our results show that continuous moderate‐level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA_Aβ3. Furthermore, noise rearing also induced a significant decrease in the level of GABA_A receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA_Aβ3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. © 2009 Wiley‐Liss, Inc.     

Electron tomography on γ‐aminobutyric acid‐ergic synapses reveals a discontinuous postsynaptic network of filaments

The regulation of synaptic strength at γ‐aminobutyric acid (GABA)‐ergic synapses is dependent on the dynamic capture, retention, and modulation of GABA A‐type receptors by cytoplasmic proteins at GABAergic postsynaptic sites. How these proteins are oriented and organized in the postsynaptic cytoplasm is not yet established. To better understand these structures and gain further insight into the mechanisms by which they regulate receptor populations at postsynaptic sites, we utilized electron tomography to examine GABAergic synapses in dissociated rat hippocampal cultures. GABAergic synapses were identified and selected for tomography by using a set of criteria derived from the structure of immunogold‐labeled GABAergic synapses. Tomography revealed a complex postsynaptic network composed of filaments that extend ～100 nm into the cytoplasm from the postsynaptic membrane. The distribution of these postsynaptic filaments was strikingly similar to that of the immunogold label for gephyrin. Filaments were interconnected through uniform patterns of contact, forming complexes composed of 2–12 filaments each. Complexes did not link to form an integrated, continuous scaffold, suggesting that GABAergic postsynaptic specializations are less rigidly organized than glutamatergic postsynaptic densities. J. Comp. Neurol. 522:921–936, 2014. © 2013 Wiley Periodicals, Inc.     

Immunocytochemical localization of the α6 subunit of the γ-aminobutyric acidA receptor in the rat nervous system

The localization in the rat central nervous system and retina of the α₆ subunit peptide of the γ-aminobutyric acid (GABA_A) receptor has been studied by light microscopy immunocytochemistry with a specific anti-α₆ antibody. The α₆ subunit was present in the granule cells of the cerebellum, the granule cells of the dorsal cochlear nucleus, axons of the olfactory nerve including the glomerular endings, layer II of the dorsal horn of the spinal cord, and in the retinal synaptic layers, particularly the inner plexiform layer. Thus, contrary to the general belief, the α₆ subunit is not exclusively localized in the granule cells of the cerebellum. It is also expressed in some sensory neurons and other neurons involved in the initial processing of sensory information. © 1996 Wiley-Liss, Inc.     

Plasticity of synaptic inhibition in mouse spinal cord lamina II neurons during early postnatal development and after inactivation of the glycine receptor α3 subunit gene

Synaptic inhibition mediated by GABA_A receptors and glycine receptors (GlyRs) in the outer laminae of the spinal cord dorsal horn efficiently filters ascending nociceptive messages, controlling pathological pain symptoms. However, although many studies have utilized transgenic models to study spinal nociceptive processing, very little is known about the development of functional inhibitory synapses onto these interneurons in mice. Here we report that most interneurons in lamina II are placed under phasic control by both GABAergic and glycinergic synapses, a number of which exhibit dual GABA/glycine co‐release. A developmental switch is also apparent: a subpopulation of lamina II interneurons controlled exclusively by either GABAergic or glycinergic synapses becomes detectable only after postnatal days 15 and 21, respectively. Using mice older than postnatal day 21, we also characterized the plastic changes in glycinergic transmission resulting from the inactivation of the GlyR α3 subunit gene, a key player in inflammatory pain pathways. This allowed us to demonstrate that synapses containing GlyR α3 contribute in large part to synaptic inhibition in lamina II. In Glra3 knockout mice, we found that synaptic currents at the remaining glycinergic synapses, containing GlyR α1, showed faster decay kinetics with unchanged amplitudes but increased frequency. These findings explain the absence of any basal nociceptive hypersensitivity in Glra3 knockout mice, as GlyR α1 is still available for mediating synaptic inhibition at lamina II synapses, but cannot be modulated by the prostaglandin–E‐prostanoid type 2 (EP2) receptor–protein kinase A signalling cascade. Our results clearly demonstrate that presynaptic GABA/glycine release properties are influenced by the nature and complexity of postsynaptic inhibitory receptor subtypes.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

γ‐Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABA_A receptor. Nineteen different subunits (α1‐6, β1‐3, γ1‐3, δ, ε, π, θ, ρ1‐3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABA_A receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure‐ and age‐characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3‐immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure‐ and age‐characteristic colocalization between subunits during development suggests differences in GABA_A receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans. J. Comp. Neurol. 524:1805–1824, 2016. © 2015 Wiley Periodicals, Inc.     

Roles for γ‐aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus

The development of the hypothalamic paraventricular nucleus (PVN) involves several factors that work together to establish a cell group that regulates neuroendocrine functions and behaviors. Several molecular markers were noted within the developing PVN, including estrogen receptors (ER), neuronal nitric oxide synthase (nNOS), and brain‐derived neurotrophic factor (BDNF). By contrast, immunoreactive γ‐aminobutyric acid (GABA) was found in cells and fibers surrounding the PVN. Two animal models were used to test the hypothesis that GABA works through GABA_A and GABA_B receptors to influence the development of the PVN. Treatment with bicuculline to decrease GABA_A receptor signaling from embryonic day (E) 10 to E17 resulted in fewer cells containing immunoreactive (ir) ERα in the region of the PVN vs. control. GABA_BR1 receptor subunit knockout mice were used to examine the PVN at P0 without GABA_B signaling. In female but not male GABA_BR1 subunit knockout mice, the positions of cells containing ir ERα shifted from medial to lateral compared with wild‐type controls, whereas the total number of ir ERα‐containing cells was unchanged. In E17 knockout mice, ir nNOS cells and fibers were spread over a greater area. There was also a significant decrease in ir BDNF in the knockout mice in a region‐dependent manner. Changes in cell position and protein expression subsequent to disruption of GABA signaling may be due, in part, to changes in nNOS and BDNF signaling. Based on the current study, the PVN can be added as another site where GABA exerts morphogenetic actions in development. J. Comp. Neurol. 518:2710–2728, 2010. © 2010 Wiley‐Liss, Inc.     

Glycine receptors in the striatum, globus pallidus, and substantia nigra of the human brain: an immunohistochemical study

Glycine receptors (GlyRs) are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian spinal cord and brain. GlyRs have four functional subunits, alpha1-3 and beta, which likely exist in heteromeric alphabeta combinations. Mutations in GlyR alpha1 and beta subunits are well known for their involvement in hyperekplexia, a paroxysmal motor disorder. In this study we present the first detailed immunohistochemical investigation at the regional, cellular, and subcellular levels of GlyRs in the human basal ganglia. The results show that GlyRs are present at the regional level in low concentrations in the striatum and globus pallidus and are present in the highest concentrations in the substantia nigra. At the cellular level, GlyRs are present only in discrete populations of neurons immunoreactive for choline acetyltransferase (ChAT), parvalbumin, and calretinin in the human striatum, on a subpopulation of parvalbumin- and calretinin-positive neurons in the globus pallidus, and in the substantia nigra GlyRs are present on approximately three-fourths of all pars compacta and one-third of all pars reticulata neurons. They also form a distinct band of immunoreactive neurons in the intermedullary layers of the globus pallidus. At the subcellular level in the substantia nigra pars reticulata (SNr), GlyRs show a localized distribution on the soma and dendrites that partially complements but does not overlap with the distribution of gamma-aminobutyric acid (GABA)A receptors. Our results demonstrate the precise cellular and subcellular localization of GlyRs in the human basal ganglia and suggest that glycinergic receptors may play an important complementary role to other inhibitory receptors in modulating cholinergic, dopaminergic, and GABAergic neuronal pathways in the basal ganglia.     

Granule cells of the internal granule layer have increased expression of GABAa receptor β2/β3 subunits

We have examined the expression of GABA_A receptor β2/β3 subunits from postnatal day 4 (P4) to P23. β2/ β3 subunits are not detected in premigratory granule cells. Expression of β2/β3 was also low in granule cells found in the internal granule layers (IGLs) of P7 and P10 cerebella. However, between P10 and P16, the levels of β2/β3 increase substantially and reach high levels at P16–23. Because granule cells continue to migrate from the external granule layer (EGL) into the IGL after P10, this increase in the number of cells and the intensity of β2/β3 expression could be the result of a second wave of granule cells expressing β2/β3 that have migrated from the EGL. To test this hypothesis, migrating granule cells after P10 were eliminated by γ-irradiation. Despite elimination of migrating granule cells, β2/3 expression remained high in the IGLs of P16 irradiated animals similar to that observed in non-irradiated controls, suggesting that the increase of β2/β3 is not due to the arrival of a new population of granule cells expressing GABA_A receptors. To explore the possibility that the increase of β2/β3 is triggered by synaptic activity, we cultured P10 cerebellar sections, free of mossy fiber inputs, for 6 days in vitro. As observed in vivo, β2/3 expression in the IGL of cultured slices continued to increase, suggesting that β2/3 expression could be triggered without synaptic inputs from mossy fibers. J. Neurosci. Res. 51:697–711, 1998. © 1998 Wiley-Liss, Inc.     

Rapid use‐dependent down‐regulation of γ‐aminobutyric acid type A receptors in rat mesencephalic trigeminal neurons

Rundown is ubiquitously seen in response to repetitive activation of receptor or ion channels as a use‐dependent down‐regulation through various mechanisms. In contrast to AMPA receptors, γ‐aminobutyric acid type A receptor (GABA_AR) are believed to display no rapid use‐dependent down‐regulation. We report here a rapid use‐dependent down‐regulation of GABA_AR in primary sensory neurons of rat mesencephalic trigeminal nucleus (MTN), which express synaptic GABA_ARs in addition to extrasynaptic ones, unlike other primary sensory neurons. When muscimol was repetitively puff‐applied to an MTN neuron every 2 min before, during, and after the muscimol bath application for 5 min, both the GABA_A responses obtained under both current‐ and voltage‐clamp conditions were almost completely depressed during the bath application. However, the former and latter GABA_A responses recovered to 26% ± 7% and 36% ± 7% of their control amplitudes, respectively, 15 min after washout of the bath‐applied muscimol. By contrast, when examined in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, together with a stringent chelation of intracellular Ca²⁺, the puff responses were almost completely recovered, whereas those were recovered to 40–60% of the control by either chelerythrine or EGTA alone. A phosphatidylinositol 3‐kinase inhibitor (PI3K), wortmannin, which blocks various signal transductions, including vesicular trafficking, significantly enhanced the rundown of the puff responses examined every 2 min. These findings indicate that the rundown of GABA_A response in MTN neurons is mediated by the use‐dependent down‐regulation of GABA_AR, which is reversed by PKC inhibition together with intracellular Ca²⁺ chelation, while being facilitated by PI3K inhibition. © 2009 Wiley‐Liss, Inc.     

Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography Changes in amyotrophic lateral sclerosis

Binding sites for the receptor ligands 3H-quinuclidinylbenzilate, 3H-alpha-bungarotoxin (3H-alpha-Btx), 3H-etorphine and 3H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distributed within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged.     

Immunocytochemical localization of the β2 subunit of the gamma-aminobutyric acidA receptor in the rat brain

An antiserum to the β₂ subunit of the rat gamma-aminobutyric acid (GABA_A) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the β₂ subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABA_A receptors. The anti-β₂ antibodies were affinity purified on immobilized β₂ intracellular loop peptide. The antibodies recognized a 55–57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABA_A receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the β₂ subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-β₂ antibody and the monoclonal antibody 62-3G1 (which recognizes both β₂ and β₃ subunits) is presented. The procedure described for generating and preparing specific anti-β₂ subunit antibodies that are valuable for immunocytochemistry could be extended to other GABA_A receptor subunits. © 1994 Wiley-Liss, Inc.     

Comparison of the regional expression of nicotinic acetylcholine receptor α7 mRNA and [125I]-α-bungarotoxin binding in human postmortem brain

Neuronal nicotinic acetylcholine receptors are expressed in the human central nervous system. A specific subtype of this receptor family, the α7 nicotinic acetylcholine receptor, is thought to be the principal α-bungarotoxin (αBTX)-binding protein in mammalian brain. Although the expression of this receptor subtype has been characterized in rat, no study has specifically compared the expression of both the α7 gene and the localization of BTX binding sites in human brain. Expression of α7 mRNA and receptor protein in human postmortem brain tissue was examined by in situ hybridization and [¹²⁵I]-α-bungarotoxin autoradiography, respectively, with particular emphasis on regions associated with sensory processing. Regions with high levels of both α7 gene expression and [¹²⁵I]-αBTX binding include the nucleus reticularis of the thalamus, the lateral and medial geniculate bodies, the basilar pontine nucleus, the horizontal limb of the diagonal band of Broca, the nucleus basalis of Meynert, and the inferior olivary nucleus. High-to-moderate levels of α7 probe hybridization were also seen in the hippocampus and the cerebral cortex; however, there was a reduced or variable degree of [¹²⁵I]-αBTX binding in these regions compared with the level of probe hybridization. In most brain regions, [¹²⁵I]-αBTX binding was localized to neuronal cell bodies similar in morphology to those that exhibited α7 hybridization, suggesting that the high-affinity [¹²⁵I]-αBTX binding sites in the human brain are likely to be principally composed of α7 receptor subtypes. J. Comp. Neurol. 387:385–398, 1997. © 1997 Wiley-Liss, Inc.     

Aspartate‐containing neurons of the brainstem and rostral spinal cord of the sea lamprey Petromyzon marinus: Distribution and comparison with γ‐aminobutyric acid

The amino acid L‐aspartate (ASP) is one of the most abundant excitatory neurotransmitters in the mammalian brain, but its distribution in other vertebrates has not yet been well characterized. We investigated the distribution of ASP in the brainstem and rostral spinal cord of the adult sea lamprey by using ASP immunohistochemistry. Our results indicate that ASP is accumulated in specific neurons, but not in glia (tanycytes). ASP‐immunoreactive neuronal populations were rather similar as the glutamatergic populations reported in the adult sea lamprey (Villar‐Cerviño et al. [2013] J Comp Neurol 521:522–557), although some important differences were noted. Characteristically, the largest reticular neurons of the lamprey brainstem (Müller cells) showed ASP immunoreactivity in perikarya and processes, in contrast to the absence or faint glutamate immunoreactivity reported in these perikarya. We also compared the distribution of ASP and γ‐aminobutyric acid (GABA) in brainstem neurons by using double immunofluorescence methods. In regions such as the midbrain tectum, dorsal isthmus, and motor nuclei, ASP and GABA immunoreactivity was mostly located in different neurons, whereas in other nuclei (torus semicircularis, octavolateralis area, parvocellular reticular formation), many of the ASP‐immunonegative neurons displayed colocalization with GABA. These results, together with those of our previous studies of colocalization of glutamate and GABA, suggest that some lamprey neurons may co‐release both excitatory and inhibitory neurotransmitters. Further investigation is needed to elucidate the pathways of uptake and release of ASP by ASP‐immunoreactive neurons. Our results indicate that ASP is a neurotransmitter in the central nervous system representative of agnathans, the earliest vertebrate group. J. Comp. Neurol. 522:1209–1231, 2014. © 2013 Wiley Periodicals, Inc.     

Altered balance of γ‐aminobutyric acidergic and glutamatergic afferent inputs in rostral ventrolateral medulla‐projecting neurons in the paraventricular nucleus of the hypothalamus of renovascular hypertensive rats

An imbalance of excitatory and inhibitory functions has been shown to contribute to numerous pathological disorders. Accumulating evidence supports the idea that a change in hypothalamic γ‐aminobutyric acid (GABA)‐ergic inhibitory and glutamatergic excitatory synaptic functions contributes to exacerbated neurohumoral drive in prevalent cardiovascular disorders, including hypertension. However, the precise underlying mechanisms and neuronal substrates are still not fully elucidated. In the present study, we combined quantitative immunohistochemistry with neuronal tract tracing to determine whether plastic remodeling of afferent GABAergic and glutamatergic inputs into identified RVLM‐projecting neurons of the hypothalamic paraventricular nucleus (PVN‐RVLM) contributes to an imbalanced excitatory/inhibitory function in renovascular hypertensive rats (RVH). Our results indicate that both GABAergic and glutamatergic innervation densities increased in oxytocin‐positive, PVN‐RVLM (OT‐PVN‐RVLM) neurons in RVH rats. Despite this concomitant increase, time‐dependent and compartment‐specific differences in the reorganization of these inputs resulted in an altered balance of excitatory/inhibitory inputs in somatic and dendritic compartments. A net predominance of excitatory over inhibitory inputs was found in OT‐PVN‐RVLM proximal dendrites. Our results indicate that, along with previously described changes in neurotransmitter release probability and postsynaptic receptor function, remodeling of GABAergic and glutamatergic afferent inputs contributes as an underlying mechanism to the altered excitatory/inhibitory balance in the PVN of hypertensive rats. J. Comp. Neurol. 518:567–585, 2010. © 2009 Wiley‐Liss, Inc.     

Distribution of immunoreactivity for the β2 and β3 subunits of the GABAA receptor in the mammalian spinal cord

The localization of GABA_A receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the β₂ and β₃ subunits of the receptor. β₂/β₃-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I–III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that β₂/β₃-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of β₂/β₃-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of β₂/β₃-subunit immunoreactivity were also detected using immunofluorescence. Focal “hot spots” correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of β₂/β₃-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane β₂/β₃ immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of “classical” (synaptic) and “nonclassical” (paracrine) actions mediated via spinal cord GABA_A receptors. The study also revealed the relative paucity of β₂/β₃-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals. © 1996 Wiley-Liss, Inc.